Clinical usefulness of urinary biomarkers for early prediction of acute kidney injury in patients undergoing transaortic valve implantation

This study aimed to reveal the clinical usefulness of urinary biomarkers for the early prediction of AKI onset after transcatheter aortic valve implantation (TAVI) (n = 173). In this study, 22 (12.7%) patients had AKI, of which 21 had mild AKI and 1 had moderate AKI. Higher levels of urinary liver-type fatty acid binding protein (L-FABP), [tissue inhibitor of metalloproteinases-2] × [insulin-like growth factor-binding protein 7], clusterin and urinary albumin before, after and 4 h after TAVI were associated with AKI onset. However, the time point of higher urinary N-acetyl-β-d-glucosaminidase levels related to AKI onset was only before TAVI. No significant differences were found in the area under the receiver-operator characteristic curves (AUC) for predicting AKI onset between urinary biomarkers before TAVI. After TAVI, the AUC (0.81) of urinary albumin was significantly higher than those of any other urinary biomarkers. The sensitivity (0.86) in urinary albumin after TAVI and specificity (0.98) in urinary L-FABP before TAVI were the highest among urinary biomarkers. In conclusion, urinary biomarkers may be clinically useful for early differentiation of patients with a higher or lower risk for AKI onset or early prediction of post-TAVI onset of AKI.

Changes in urinary biomarkers in the AKI and non-AKI groups are shown in Fig. 1.In the AKI group, urinary levels of L-FABP significantly increased post-operation (P < 0.01), 4-h post-operation (P < 0.05) and on PODs 1 (P < 0.05) and 3 (P < 0.01) compared with those pre-operation (Fig. 1).In the non-AKI group, urinary L-FABP levels significantly increased post-operation, 4-h post-operation and on PODs 1, 2 and 3 compared with those pre-operation (P < 0.01, Fig. 1).Urinary L-FABP levels were significantly higher at all measurement points in the AKI group compared with those in the non-AKI group (P < 0.05, Fig. 1).
In the AKI group, urinary levels of clusterin significantly increased post-operation (P < 0.01), 4-h post-operation (P < 0.01) and on POD 1 (P < 0.05) compared with those pre-operation (Fig. 1).In the non-AKI group, urinary clusterin levels significantly increased post-operation, 4-h post-operation and on PODs 1, 2 and 3 compared with those pre-operation (P < 0.01, Fig. 1).Urinary clusterin levels were significantly higher in the AKI group than in the non-AKI group at all other points, except that on POD 3 (P < 0.05, Fig. 1).
In the AKI group, urinary levels of NAG significantly increased on PODs 2 (P < 0.01) and 3 (P < 0.01) compared with those pre-operation (Fig. 1).In the non-AKI group, urinary NAG levels significantly increased 4-h post-operation and on PODs 1, 2 and 3 compared with those pre-operation (P < 0.01, Fig. 1).Urinary NAG levels pre-operation and on POD 2 were significantly higher in the AKI group than in the non-AKI group (P < 0.05, Fig. 1).

Binomial logistic regression analysis for predicting post-TAVI onset of AKI
In this study, to reveal whether urinary biomarkers at the early phase (pre-operation, post-operation and 4-h post-operation) were associated with AKI onset, binomial logistic regression analysis was performed using ejection fraction, diabetes mellitus and CKD as explanatory variables because these factors were significantly observed in the AKI group compared with the non-AKI group (Table 3).All urinary biomarkers pre-operation, post-operation and 4-h post-operation, except for urinary NAG postoperation, were significantly associated with AKI onset in the unadjusted analysis and after adjustment for diabetes mellitus and CKD (Table 3).Furthermore, all urinary biomarkers pre-operation, post-operation and 4-h post-operation, except for urinary NAG post-operation and 4-h post-operation, independently predicted AKI onset after further adjustment for ejection fraction (Table 3).

Receiver-operating characteristic (ROC) curves analysis of urinary biomarkers and SCr for predicting the post-TAVI onset of AKI
Area under the ROC curves (AUCs) of each urinary biomarker for predicting the post-TAVI onset of AKI are shown in Table 4.Although no significant differences in AUC levels were found between urinary biomarkers pre-operation, the AUC level of urinary albumin (0.81) post-operation was significantly higher than any other urinary biomarkers at the same time point (Table 4).Furthermore, post-operation, the AUC level of urinary L-FABP (0.65) was significantly higher than that of urinary NAG (0.41) (Table 4).Moreover, 4-h post-operation, Table 3. Results of binomial logistic regression analyses for AKI in the TAVI.*P < 0.05.AKI acute kidney injury, TAVI Transcatheter Aortic Valve Implantation, DM Diabetes Mellitus, CKD Chronic kidney disease, EF Ejection fraction, OR odds ratio, CI confidence interval, L-FABP liver-type fatty acid binding protein, TIMP-2 tissue inhibitor of metalloproteinases-2, IGFBP7 insulin-like growth factor-binding protein 7, NAG N-acetyl-βd-glucosamin. www.nature.com/scientificreports/ the AUC level of urinary clusterin (0.76) was significantly higher than that of urinary L-FABP (0.63) (Table 4).
Each cutoff level of urinary parameters for predicting AKI onset is shown in Table 5.The sensitivity (0.86) of the cutoff value (151.4 mg/g.cr) in urinary albumin post-operation and the specificity (0.98) of the cutoff value (35.3 µg/g.cr) in urinary L-FABP pre-operation were the highest among those of all urinary biomarkers (Table 5).

Discussion
This study showed that post-TAVI onset of AKI was significantly observed in older patients with decreased ejection fraction, diabetes mellitus or severe CKD.In addition, higher levels of peri-operative urinary L-FABP, [TIMP-2] × [IGFBP7], clusterin, NAG and albumin at the early phase were significantly associated with the post-TAVI onset of AKI.While the AUC levels in each urinary biomarker were not significantly different preoperation, the AUC in urinary albumin post-operation showed the highest level among all urinary biomarkers measured at the same time point.The cutoff value of urinary albumin post-operation showed the highest sensitivity, whereas that of urinary L-FABP pre-operation showed the highest specificity.Urinary biomarkers may be useful for early differentiation of patients with a higher or lower risk for AKI onset or early prediction of post-TAVI onset of AKI.
The pathophysiology of post-TAVI AKI has not been sufficiently clarified and can be caused by various factors.Previously, the higher volume of the contrast medium injected during TAVI was reported to be a risk factor for contrast-induced nephropathy (CIN) 9 .However, the present study significantly showed a lower volume of the contrast medium in the AKI group than in the non-AKI group.A recent large-scale study did not show a causal relationship between the administration of the contrast medium and AKI 10,11 .On the contrary, preexisting renal insufficiency is the most important risk factor for CIN 12 .In the present study, impaired preoperative renal function was significantly observed in the AKI group compared with the non-AKI group, and the levels of urinary biomarkers that reflect tubular damage were significantly higher in the AKI group than in the non-AKI group before TAVI, showing that the kidneys of patients with AKI may be the most vulnerable to an even lower volume of contrast medium.Furthermore, the levels of urinary L-FABP, which reflects renal ischaemia 13,14 , significantly increased after TAVI than before TAVI in both AKI and non-AKI groups, and levels of urinary L-FABP remained significantly in the AKI group than in the non-AKI group during the peri-operative period of TAVI, suggesting that TAVI caused renal ischaemia and that more severe renal ischaemia may be provoked by TAVI in the kidneys of patients with AKI.A contrast medium induces tubular damage by not only direct toxic effects on tubular cells but also renal ischaemia brought by vasoconstriction 15 .In this study, the contrast medium injected during TAVI may contribute to the induction of post-TAVI AKI in patients with preexisting renal dysfunction and tubular damage.
L-FABP is localised in the cytoplasm of the proximal tubule and is excreted into the urine by ischaemic stress on the tubule before the tissue damage progresses 14 .The clinical usefulness of urinary L-FABP in predicting AKI was reported in various pathophysiology [16][17][18] , which was supported by the results of the present study.In addition, Table 5. Biomarkers levels predictive of AKI in the TAVI.AKI acute kidney injury, TAVI transcatheter aortic valve implantation, pre-op preoperation, post-op immediate post-operation, 4 h post-op 4 h after surgery, POD post-operative day, L-FABP liver-type fatty acid binding protein, TIMP-2 tissue inhibitor of metalloproteinases-2, IGFBP7 insulin-like growth factor-binding protein 7, NAG N-acetyl-β-dglucosaminidase. the specificity of urinary L-FABP before TAVI was the highest among those in all urinary biomarkers.Urinary L-FABP may have potential for the differentiation of patients without post-TAVI onset of AKI in addition to prediction of early post-TAVI onset of AKI.TIMP-2 and IGFBP7 are related to the induction of G1 cell-cycle arrest 19 .Their expressions are up-regulated in proximal cells by tubular injury; thereafter, they are excreted from the tubules into the urine 19 .Recently, a multicenter study reported the discovery and validation of two novel biomarkers of AKI, i.e. urinary TIMP-2 and IGFBP7 20 , in patients with critical illness 19 .Urinary [TIMP-2] × [IGFBP7] in patients with critical illness after ICU admission was shown to be useful for predicting the development of moderate or severe AKI (KDIGO stages 2-3) within 12 h 19 .In the present study, urinary [TIMP-2] × [IGFBP7] levels significantly increased after TAVI compared with before TAVI in both AKI and non-AKI groups and was significantly higher in the AKI group than in the non-AKI group at the late phase after TAVI.As the G1 cell-cycle arrest in the proximal tubule plays a renal protective role against AKI 21 , the G1 cell-cycle arrest in the tubules was strongly induced in the kidneys of patients with post-TAVI AKI for the prevention of tubular damage.On the contrary, although higher levels of urinary [TIMP-2] × [IGFBP7] after TAVI in addition to those before TAVI were associated with AKI onset, our study did not show a significant difference in urinary [TIMP-2] × [IGFBP7] levels between the AKI and non-AKI groups at the early phase after TAVI.Therefore, urinary [TIMP-2] × [IGFBP7] may be useful in differentiating patients at high risk for AKI before TAVI.
Clusterin is a 5-80 kDa heterodimeric glycoprotein that was reported to regulate apoptosis, lipid transport, cell interactions and complement 22 .Its expression has been reported to be up-regulated in proximal tubular cells depolarised after ischaemia or renal damage and reflects tissue fibrosis 23,24 .Levels of urinary clusterin were significantly higher in the AKI group than in the non-AKI group at both early and late phases, increased significantly after TAVI than before TAVI in both AK and non-AKI groups, and were significantly associated with AKI onset.These results showed the possibility that urinary clusterin may be useful in differentiating patients at high risk of AKI onset before TAVI and predicting early the post-TAVI onset of AKI.
Urinary NAG is a conventional biomarker that reflects proximal tubular damage.In this study, urinary NAG levels significantly increased from 4-h post-operation compared with pre-operation in the non-AKI group but not in the AKI group.On the contrary, significantly higher urinary NAG levels in the AKI group than in the non-AKI group were observed pre-operation but not at the early phase after TAVI.Furthermore, higher levels of NAG before TAVI were associated with AKI onset but not after TAVI.From these results, urinary NAG before TAVI may be useful in differentiating patients at high risk of AKI onset.
Urinary albumin is widely recognized to reflect tubular injury in addition to glomerular damage 25 .The higher levels of urinary albumin before TAVI were reported to be associated with not only AKI onset but also all-cause death and heart failure readmission and may be noticed for predicting the prognosis after TAVI 26 .This study showed the retention of significantly higher urinary albumin levels in the AKI group than in the non-AKI group during the peri-operative period of TAVI and a significant increase in urinary albumin levels at the early phase after TAVI compared with pre-operation in both AKI and non-AKI groups.Urinary albumin levels preoperation and early phase after TAVI were significantly associated with AKI onset.Furthermore, regarding the time point of immediately after TAVI, the AUC of urinary albumin for predicting AKI onset was the highest among AUCs of all urinary biomarkers.Tubular injury causes the impairment of peri-tubular capillary, which leads to glomerular damage that is located upstream of the peri-tubular capillary.The procedure of TAVI is considered to provoke tubular and glomerular damage.As urinary excretion of albumin is increased by glomerular and tubular damage, urinary albumin may be a prominent marker for predicting AKI onset.This study indicated that compared with other biomarkers, measuring urinary albumin immediately after TAVI is more useful for predicting AKI onset peri-operatively.
The present study showed that the sensitivity (0.86) of the cut-off value (151.4 mg/g.cr) in urinary albumin post-operation and the specificity (0.98) of the cut-off value (35.3 µg/g.cr) in urinary L-FABP pre-operation were the highest among those of all urinary biomarkers.Although this point is needed to be reconfirmed in multicenter studies, measurement of urinary L-FABP before the procedure of TAVI may be useful for discrimination of patients without post-TAVI onset of AKI, whereas measurement of urinary albumin after TAVI may be superior for early predicting the post-TAVI onset of AKI.
We considered that urinary biomarkers which reflect the pathophysiology of post-TAVI AKI may be useful for early prediction of AKI due to TAVI.Contrast medium, which induces proximal tubular injury via both direct toxic actions and renal ischemia, is an important cause for post-TAVI AKI as described above.Therefore, we selected urinary NAG which is increased by construction injury of proximal tubules, and both urinary L-FABP and clusterin which reflect proximal tubular ischemia.In addition, as renal ischemia induces cell cycle arrest and both TIMP-2 and IGFBP7 are related to the cell cycle arrest, urinary [TIMP-2] × [IGFBP7] was measured.Furthermore, clinical impact of urinary albumin on the prognosis after TAVI was recently reported 26 and urinary albumin was added.On the other hand, both KIM-1 and NGAL have been widely studied and are well-known tubular biomarkers.However, the relationship between urinary KIM-1 and renal ischemia was not clear.Urinary NGAL was reported to be increased by inflammation 27 .We did not determine whether increased urinary NGAL levels after TAVI was due to AKI or infection brought by procedure of TAVI.Thus, urinary KIM-1 and NGAL were not evaluated in the present study.
This study has some critical limitations.First, this was a single-centre study, and the number of patients with AKI was small, which lead to the limited number of variables included in the logistic regression analysis.Second, significant differences were found in the prevalence of CKD, each urinary biomarker level and renal function before TAVI between the AKI and non-AKI groups.The movements of each urinary biomarker after TAVI may be influenced by the presence of CKD or renal dysfunction before TAVI.Third, as albumin administration during the operation of TAVI was performed in the present study, there was a possibility that the administered albumin had an effect on urinary albumin levels.However, urinary albumin levels in the AKI group at all time points were not significantly different between the patients with and the without the albumin administration (Supplementary Fig. S1).Finally, the severity of AKI in nearly all patients with AKI was mild, and the clinical utility of urinary biomarkers for predicting the onset of moderate or severe AKI was not investigated.
In conclusion, urinary biomarkers measured in this study may be clinically useful for early differentiation of patients with a higher or lower risk for AKI onset or early prediction of post-TAVI onset of AKI.In the future, a multicenter study involving more severe AKI cases due to TAVI is needed to reveal the clinical utility and superiority of each urinary biomarker.

Methods
The study protocol was approved by the Institutional Review Board of St. Marianna University School of Medicine (No. 3049) and registered in the UMIN Clinical Data Registry (ID 000020262).Written informed consent was obtained from all the enrolled patients.The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors' institutional review board or equivalent committee.

Study design
This prospective study was conducted in a university hospital.As contrast medium is one of causes which induces post-TAVI AKI, the sample size calculation was based on the previous study which showed that urinary L-FABP before injection of the contrast medium was useful for early predicting the contrast medium-induced nephropathy (CIN) 28 .The mean difference of urinary L-FABP before the injection of contrast medium between the CIN and non-CIN groups was 11.1 with a larger standard deviation of 12.8.The sample size calculation (α = 5%, power of 80%) resulted in 14 patients in the AKI group and 56 patients in the non-AKI group.In the present study, consecutive patients scheduled for TAVI (n = 173) between December 2016 and June 2020 were enrolled.Each patient's surgeon chose between balloon expansion and self-expansion by considering the type of the aortic valve, in the absence of any concern regarding the patient's tolerance for study procedures.Patients undergoing dialysis or requiring emergency operation were excluded from the study.
All patients who underwent TAVI received either a balloon-expandable Edwards SAPIEN transcatheter heart valve (Edwards Lifesciences, Irvine, CA, USA) or a self-expandable Medtronic Core Valve (Medtronic Incorporation, Minneapolis, MN, USA) for clinical indications.Procedures were performed under general anesthesia with endotracheal intubation and insert transesophageal echocardiography for circulation management.TAVI devices were delivered through the femoral approach.

Anesthesia
No premedication was given, and general anesthesia was induced with remifentanil and propofol.Tracheal intubation was facilitated with rocuronium, general anesthesia was maintained with sevoflurane in an air-oxygen mixture, and remifentanil, and noradrenaline was infused continuously during the operation in all patients.
If needed, nicorandil and/or carperitide were infused continuously during the operation in some groups.For transfusion, colloidal fluid, Ringer's acetate, maltose Ringer, concentrated red blood cell fluid, fresh-frozen plasma, and albumin were used.

Sample collection
Urine samples (10 mL) were obtained preoperatively on admission (pre-op), immediately after returning to the ward after the operation (post-op), 4 h after returning to the ward (4-h post-op), and POD 1, 2, and 3, respectively, for the measurement of urinary L-FABP, [TIMP-2] × [IGFBP7], clusterin, NAG, and albumin.Urine samples were centrifuged at 1000×g for 5 min at 4 °C and stored at − 80 °C until analysis.In addition, serum samples were obtained preoperatively on admission, immediately after returning to the ward after the operation, and on PODs 1, 2, 3, 4, 5, 6, and 7 for the measurement of SCr.

Clinical monitoring of patients with TAVI
The timing for the diagnosis of AKI was extended from 72 h to 7 days.During the first 48-h postoperative period, patients were monitored for AKI, which was defined according to the VARC-2 criteria 3 .The staging of postoperative AKI was made as follows: stage 1, an increase in SCr to 150%-199% (1.5-1.99 × increase compared with baseline), increase of ≥ 0.3 mg/dL (≥ 26.4 mmol/L), or urine output < 0.5 mL/kg/h for 6-12 h; stage 2, an increase in SCr to 200%-299% (2.0-2.99 × increase compared with baseline) or urine output < 0.5 mL/kg/h for 12-24 h; stage 3, induction of renal replacement therapy.
At the start of this study, the estimated glomerular filtration rate (eGFR) was also monitored, which was calculated according to the Japanese-coefficient modified Chronic Kidney Disease Epidemiology Collaboration equation 29 :